Kathrin Mandery

Influence of the flavonoids apigenin, kaempferol, and quercetin on the function of organic anion transporting polypeptides 1A2 and 2B1.

OATP1A2 and OATP2B1 are uptake transporters of the human organic anion transporting polypeptide (OATP) family with a broad substrate spectrum including several endogenous compounds as well as drugs such as the antihistaminic drug fexofenadine and HMG-CoA reductase inhibitors. Both transporters are localized in the apical membrane of human enterocytes. Flavonoids, abundantly occurring in plants, have previously been shown to interact with drug metabolizing enzymes and transporters. However, the impact of flavonoids on OATP1A2 and OATP2B1 transport function has not been analyzed in detail. Therefore, HEK293 cell lines stably expressing OATP1A2 and OATP2B1 were used to investigate the influence of the Ginkgo flavonoids apigenin, kaempferol, and quercetin on the transport activity of OATP1A2 and OATP2B1. K(i) values of all three flavonoids determined from Dixon plot analyses using BSP as substrate indicated a competitive inhibition with quercetin as the most potent inhibitor of OATP1A2 (22.0μM) and OATP2B1 (8.7μM) followed by kaempferol (OATP1A2: 25.2μM, OATP2B1: 15.1μM) and apigenin (OATP1A2: 32.4μM OATP2B1: 20.8μM). Apigenin, kaempferol, and quercetin led to a concentration-dependent decrease of the OATP1A2-mediated fexofenadine transport with IC(50) values of 4.3μM, 12.0μM, and 12.6μM, respectively. The OATP1A2- and OATP2B1-mediated transport of atorvastatin was also efficiently inhibited by apigenin (IC(50) for OATP1A2: 9.3μM, OATP2B1: 13.9μM), kaempferol (IC(50) for OATP1A2: 37.3μM, OATP2B1: 20.7μM) and quercetin (IC(50) for OATP1A2: 13.5μM, OATP2B1: 14.1μM). These data indicate that modification of OATP1A2 and OATP2B1 transport activity by apigenin, kaempferol, and quercetin may be a mechanism for food-drug or drug-drug interactions in humans.

Role of organic cation transporter OCT2 and multidrug and toxin extrusion proteins MATE1 and MATE2-K for transport and drug interactions of the antiviral lamivudine

The antiviral lamivudine is cleared predominantly by the kidney with a relevant contribution of renal tubular secretion. It is not clear which drug transporters mediate lamivudine renal secretion. Our aim was to investigate lamivudine as substrate of the renal drug transporters organic cation transporter 2 (OCT2) and multidrug and toxin extrusion proteins MATE1 and MATE2-K. Uptake experiments were performed in OCT2, MATE1, or MATE2-K single-transfected human embryonic kidney 293 (HEK) cells. Transcellular transport experiments were performed in OCT2 and/or MATE1 single- or double-transfected Madin-Darby canine kidney II (MDCK) cells grown on transwell filters. Lamivudine uptake was significantly increased in HEK-OCT2, HEK-MATE1, and HEK-MATE2-K cells compared to control cells. In transcellular experiments, OCT2 located in the basolateral membrane had no effect on transcellular lamivudine transport. MATE1 located in the apical membrane decreased intracellular concentrations and increased transcellular transport of lamivudine from the basal to the apical compartment. MATE1- or MATE2-K-mediated transport was increased by an oppositely directed pH gradient. Several simultaneously administered drugs inhibited OCT2- or MATE2-K-mediated lamivudine uptake. The strongest inhibitors were carvedilol for OCT2 and trimethoprim for MATE2-K (inhibition by 96.3 and 83.7% at 15 μM, respectively, p<0.001). Trimethoprim inhibited OCT2- and MATE2-K-mediated lamivudine uptake with IC₅₀ values of 13.2 and 0.66 μM, respectively. Transcellular lamivudine transport in OCT2-MATE1 double-transfected cells was inhibited by trimethoprim with an IC₅₀ value of 6.9 μM. Lamivudine is a substrate of renal drug transporters OCT2, MATE1, and MATE2-K. Concomitant administration of drugs that inhibit these transporters could decrease renal clearance of lamivudine.

Inhibition of hepatic uptake transporters by flavonoids

Members of the human SLC superfamily such as organic anion transporting polypeptide 1B1 (OATP1B1), OATP1B3, and organic cation transporter 1 (OCT1) are drug uptake transporters that are localised on the basolateral membrane of hepatocytes mediating the uptake of drugs such as atorvastatin and metformin into hepatocytes. Ingredients of food such as flavonoids influence the effects of drugs, e.g. by inhibition of drug transporters. Therefore, we investigated the impact of the Ginkgo biloba flavonoids apigenin, kaempferol, and quercetin, and the grapefruit flavonoids naringenin, naringin, and rutin on the OATP1B1, OATP1B3, and OCT1 transport activity. Transporter expressing HEK293 cell lines were used with [3H]sulfobromophthalein ([3H]BSP) as substrate for OATP1B1 and OATP1B3, [3H]atorvastatin as substrate for OATP1B1, and [3H]1-methyl-4-phenylpyridinium ([3H]MPP(+)) as substrate for OCT1. The G. biloba flavonoids showed a competitive inhibition of the OATP1B1- and OATP1B3-mediated [3H]BSP and the OATP1B1-mediated [3H]atorvastatin uptake. Quercetin was the most potent inhibitor of the OATP1B1- and OATP1B3-mediated [3H]BSP transport with K(i)-values of 8.8±0.8μM and 7.8±1.7μM, respectively. For the inhibition of the OATP1B1-mediated [3H]atorvastatin transport, apigenin was the most potent inhibitor with a K(i) value of 0.6±0.2μM. Among the grapefruit flavonoids, naringenin was the most potent inhibitor of the OATP1B1- and OATP1B3-mediated [3H]BSP transport with IC(50)-values of 81.6±1.1μM and 101.1±1.1μM, respectively. All investigated flavonoids showed no significant inhibition of the OCT1-mediated [3H]MPP(+) uptake. Taken together, these in vitro studies showed that the investigated flavonoids inhibit the OATP1B1- and OATP1B3-mediated drug transport, which could be a mechanism for food-drug interactions in humans.

The prostaglandin transporter OATP2A1 is expressed in human ocular tissues and transports the antiglaucoma prostanoid latanoprost.

PURPOSE Latanoprost, a prostaglandin F(2alpha) analogue, has become one of the most widely used medications for the treatment of glaucoma. The authors hypothesized that organic anion transporting polypeptides (OATPs) are responsible for the uptake of latanoprost into ocular tissues and, hence, that they contribute to the interindividual differences in drug concentrations and effects. METHODS Expression of prostaglandin (PG) transporters (OATP2A1, OATP2B1) in human ocular tissues was determined using real-time RT-PCR and immunofluorescence. The inhibitory interactions between latanoprost and its active metabolite (the free acid) and the uptake of prototypical substrates (PGE(2) and bromosulfophthalein) were tested in stably transfected human embryonic kidney cells overexpressing either OATP2A1 or OATP2B1. These cells were also used to investigate whether latanoprost and latanoprost acid are substrates of OATP2A1 or OATP2B1. RESULTS OATP2A1 and OATP2B1 mRNA expression was highest in the choroid/retinal pigment epithelium (RPE) complex and ciliary body. OATP2A1 protein expression was most prominent in the RPE and in epithelial and endothelial cell layers of anterior segment tissues, such as cornea, conjunctiva, iris, and ciliary body, whereas OATP2B1 protein was additionally expressed in trabecular meshwork, Schlemm canal, and choroidal vasculature. Latanoprost and latanoprost acid significantly inhibited both OATP2A1 and OATP2B1. Uptake experiments demonstrated that latanoprost acid is effectively transported by OATP2A1 (affinity constant [K(m)], 5.4 microM; maximum uptake rate [V(max)], 21.5 pmol/mg protein/min) and less effectively by OATP2B1. CONCLUSIONS The results presented herein suggest that at least OATP2A1 plays a role in the intraocular disposition of the therapeutically used prostanoid latanoprost.

Interaction of innovative small molecule drugs used for cancer therapy with drug transporters

Multiple new small molecules such as tyrosine kinase, mammalian target of rapamycin (mTOR) and proteasome inhibitors have been approved in the last decade and are a considerable progress for cancer therapy. Drug transporters are important determinants of drug concentrations in the systemic circulation. Moreover, expression of drug transporters in blood–tissue barriers (e.g. blood–brain barrier) can limit access of small molecules to the tumour (e.g. brain tumour). Finally, transporter expression and (up)regulation in the tumour itself is known to affect local drug concentrations in the tumour tissue contributing to multidrug resistance observed for multiple anticancer agents. This review summarizes the current knowledge on: (i) small molecules as substrates of uptake and efflux transporters; (ii) the impact of transporter deficiency in knockout mouse models on plasma and tissue concentrations; (iii) small molecules as inhibitors of uptake and efflux transporters with possible consequences for drug–drug interactions and the reversal of multidrug resistance; and (iv) on clinical studies investigating the association of polymorphisms in genes encoding drug transporters with pharmacokinetics, outcome and toxicity during treatment with the small molecules.

Influence of Cyclooxygenase Inhibitors on the Function of the Prostaglandin Transporter Organic Anion-Transporting Polypeptide 2A1 Expressed in Human Gastroduodenal Mucosa

The human organic anion-transporting polypeptide 2A1 (OATP2A1) is a prostaglandin transporter expressed in several tissues and plays an important role for local distribution of prostaglandins, which contribute to the integrity of gastric mucosa. Blockade of prostaglandin pathways by cyclooxygenase (COX) inhibitors has been associated with serious side effects such as gastrointestinal ulceration and bleeding. However, little is known regarding OATP2A1 expression in the upper gastrointestinal tract and the potential impact of cyclooxygenase inhibitors on OATP2A1 function. We first investigated the expression of OATP2A1 mRNA and protein in human gastroduodenal mucosa using human biopsy specimens obtained from antrum, corpus, and duodenum. The results indicate that OATP2A1 is expressed in the neck region and deep pyloric glands of antrum and in parietal cells of gastric corpus. Second, we examined various COX inhibitors for their effects on OATP2A1 transporter activity. Using HEK293 cells expressing OATP2A1, we found that diclofenac and lumiracoxib are potent inhibitors of OATP2A1-mediated transport of prostaglandin (PG) E2 with IC50 values of 6.2 ± 1.2 and 3.1 ± 1.2 μM. In contrast, indomethacin, ketoprofen, and naproxen led to significant stimulation of OATP2A1-mediated PGE2 transport by 162.7 ± 13.9, 77.2 ± 3.6, and 32.3 ± 4.9%, respectively. Taken together, our results suggest that various clinically used COX inhibitors have differential impact on the function of the prostaglandin transporter OATP2A1 in human stomach and that these effects may contribute to differences in the gastrointestinal side effects of COX inhibitors.

Relevance of conserved lysine and arginine residues in transmembrane helices for the transport activity of organic anion transporting polypeptide 1B3

Background and purpose:  Organic anion transporting polypeptide 1B3 (OATP1B3) (SLCO1B3) mediates the uptake of endogenous substrates (e.g. estrone‐3‐sulphate) and drugs (e.g. pravastatin) from blood into hepatocytes. Structure‐based modelling of OATP1B3 suggested that a pore with a positive electrostatic potential contributes to the transport mechanism. Therefore, we investigated the role of conserved positively charged amino acids for OATP1B3‐mediated uptake of sulphobromophthalein (BSP) and pravastatin.

Role of Organic Anion-Transporting Polypeptides for Cellular Mesalazine (5-Aminosalicylic Acid) Uptake

The therapeutic effects and metabolism of mesalazine (5-aminosalicylic acid) in patients with inflammatory bowel disease require intracellular accumulation of the drug in intestinal epithelial cells and hepatocytes. The molecular mechanisms of mesalazine uptake into cells have not been characterized so far. Using human embryonic kidney cells stably expressing uptake transporters of the organic anion-transporting polypeptide (OATP) family, which are expressed in human intestine and/or liver, we found that mesalazine uptake is mediated by OATP1B1, OATP1B3, and OATP2B1 but not by OATP1A2 and OATP4A1. Moreover, genetic variations (*1b, *5, *15) in the SLCO1B1 gene encoding OATP1B1 reduced the Km value for mesalazine uptake from 55.1 to 16.3, 24.3, and 32.4 μM, respectively, and the respective Vmax values. Finally, budesonide, cyclosporine, and rifampin were identified as inhibitors of OATP1B1-, OATP1B3-, and OATP2B1-meditated mesalazine uptake. These in vitro data indicate that OATP-mediated uptake and its modification by genetic factors and comedications may play a role for mesalazine effects.

Organic anion transporting polypeptides and organic cation transporter 1 contribute to the cellular uptake of the flavonoid quercetin

Flavonoids such as quercetin and kaempferol mediate several health protective effects, e.g., anticancer effects. They are inhibitors of organic anion transporting polypeptides (OATP) and organic cation transporters (e.g., OCT2). However, little is known whether such transporters contribute to the cellular uptake of flavonoids. Therefore, we investigated the cellular uptake of kaempferol and quercetin using HEK293 cell lines stably expressing different human OATPs or OCT1. Kaempferol was not a substrate of any of the investigated transporters (OATP1A2, OATP1B1, OATP1B3, OATP2A1, OATP2B1, OATP3A1, OATP4A1, OATP5A1, and OCT1). Quercetin showed a significantly higher uptake into the HEK293-OATP1A2, HEK293-OATP2A1, HEK293-OATP2B1, and HEK293-OCT1 cells compared to control cells. The OATP1A2-, OATP2B1-, and OCT1-mediated quercetin uptake was inhibited by known inhibitors such as naringin, cyclosporin A, and quinidine, respectively. The cellular accumulation of quercetin into HEK293-OATP2A1 cells was not inhibited by prostaglandin E2 and diclofenac. The ionophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) reduced the net uptake of quercetin by increasing the uptake in the HEK293-control cells and causing no significant change in the HEK293-OATP2B1 cells indicating that quercetin follows the FCCP-driven proton flux through the plasma membrane. In addition to passive diffusion, the SLC transporters OATP1A2, OATP2B1, and OCT1 contribute to cellular accumulation of quercetin.

Functional and Structural Relevance of Conserved Positively Charged Lysine Residues in Organic Anion Transporting Polypeptide 1B3

The human organic anion transporting polypeptide 1B3 (OATP1B3), located in the basolateral membrane of hepatocytes, mediates the uptake of endogenous substrates such as taurocholate and drugs from blood into hepatocytes. The transport activity of OATP1B3 is influenced by positively charged amino acids, which are facing the central pore. Molecular modeling was performed to select conserved positively charged amino acids, which may influence transport activity and anchoring of OATP1B3 in the plasma membrane. The modeling revealed that Lys361 faces the pore, and Lys399 is oriented to the plasma membrane. Therefore, the mutants L361>A, L361>R, L399>A, and L399>R were generated using site-directed mutagenesis to investigate the impact of the positive charges on transport activity and anchoring in the membrane. Transport kinetic analyses for the substrates sulfobromophthalein and taurocholate showed a loss of function for the L361>A mutant, whereas the transport activity was maintained by the L361>R mutant, indicating that the positive charge at position 361 is important for transport activity of OATP1B3. Comparative modeling with OATP1A2 and OATP2B1 revealed that the pore size around this lysine residue is larger in OATP1A2 and smaller in OATP2B1 compared with OATP1B3, which could be related to the respective substrate spectra. Cell surface expression of L399>A and L399>R was decreased to 16 and 72% compared with wild-type OATP1B3 (p < 0.001), respectively, indicating that the positive charge of lysine at position 399 is necessary for an unimpaired cell surface expression. Furthermore, we provide a summary of amino acids, which influence the transport activity of OATP1B3.